Evaluation And Optimization Of Isolation Of Non-O157 Shiga Toxin-producing Escherichia Coli

Shiga toxin-producing Escherichia coli(STEC)are globally important causes of food-borne disease,which can cause a variety of clinical outcomes ranging from diarrhea to hemolytic uremic syndrome(HUS).Ruminants are the dominant reservoir of STEC.Among more than 400 serotypes that have been discovered,the 0157:H7 serotype related to severe disease outbreak has attracted wide attention.But,in recent years,non-0157 STEC has played an important role in disease epidemic.To understand the prevalence and characteristics of non-0157 Shiga toxin-producing E.coli isolates in China,the O serogroups and H-antigen types were determined,and stx1,stx2 and eae genes were screened by PCR.Among 476 non-0157 STEC isolates,174 serotypes were detected.02:H45,08:H16 and 02:H32 were dominant serotypes.The positive rate of stx1,stx2,stx1+stx2 and eae genes were 25.84%,64.71%,9.45%,and 6.93%respectively.Thirty three isolates harbored both stx and eae genes,which mainly belonged to O26,O103 or O111 serogroups.At present,the selective isolation method of 0157 STEC has been well-established,by using agar media such as CT-MAC based on fermentation of specific carbohydrates and/or resistance to additives,while the isolation of non-0157 STEC remains a major challenge.In the present study,476 non-0157 STEC strains representing 94 different O serogroups were used to evaluate tellurite resistance and the performance of 12 different chromogenic agars.Of 476 strains,only 108 strains showed the minimal inhibitory concentration(MIC)values for potassium tellurite being higher than 4 ?g/ml,and 96 strains harbored intact ter genes cluster.The presence of ter genes was correlated with tellurite resistance.Six commercial chromogenic agars TBX,MAC,SMAC,Rainbow(?)Agar 0157,CHROMagarTM ECC and Fluorocult(?)O157:H7 supported the growth of all strains.However,CT-SMAC,CHROMagarrTM 0157 and CHROMagarTM STEC agars exhibited 12.2%,31.1%and 38.0%of growth inhibition,respectively.Furthermore,4.6%,33.2%and 45.0%strains were inhibited on RBA-USDA,RBA-NT,and BCM 0157:H7 agar media.Variations in tellurite resistance and colony appearance might result in discrepant performance of non-0157 STEC recovery from different chromogenic agars.The prevalence and characteristics of STEC isolates from fecal samples collected from beef cattle in a farm located in Sichuan province were analyzed.The positive rate of stx gene was 90.00%by real-time PCR.Of 120 fecal samples,126 STEC isolates were recovered from 87 samples(72.5%).32,31,91 and 73 strains were isolated from CHROMagarTM ECC,MAC,RBA-NT and CHROMagarTM STEC,respectively.In total,17 different O:H serotypes were identified among 126 isolates.The predominant were serotypes O84:H2,O81:H31,ONT:H8 and O5(O70):H31.Of all isolates,24.60%were stx1 positive only,which belonged to stx1a subtype;35.71%were stx2 positive and were genotyped into three subtypes(stx2a,stx2c or stxd);and 39.68%were both stx1 and stx2 positive.The positive rate of virulence factors iha,ehxA,espP,saa,eae,paa,subA,cnfl and cnf2 were 99.21%,88.10%,88.10%,58.73%,29.37%,23.81%,18.25%,0.8%,and 0.8%,respectively.However,toxB,efal and astA were all absent.The isolates were separated into 49 different patterns by pulsed-field gel electrophoresis(PFGE).The investigation of the occurrence and characteristics of STEC strains isolated from beef cattle revealed that cattles are the important hosts of STEC with diverse serotypes and genotypes.Based on our results,we propose that using inclusive agars or less selective agar in combination with highly selective agar should be suggested to recover most non-0157 STEC strains,which would increase the probability of recovering STECs from complex background microflora.Robust and reliable detection of these organisms will assist in ameliorating surveillance activities.This may consequently reduce the non-0157 STEC disease burden and prevent foodborne outbreaks associated with these pathogens.